In recent years, the public has become acutely aware of the environmental hazards and mammalian toxicity associated with the use of synthetic insecticides. As a result, the use of these insecticides has been rapidly declining. However, the need for effective insect control has not changed. This has prompted researchers to develop novel methods of insect control.
The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis (hereinafter B.t.). This bacterial agent is used to control a variety of leaf-eating caterpillars, Japanese beetles and mosquitos. U.S. Pat. No. 4,797,279 issued Jan. 10, 1989 to Karamata, et al., discloses hybrid bacterial cells comprising the gene coding for B.t. kurstaki delta-endotoxin and the gene coding for B.t. tenebrionis delta-endotoxin and their preparation. The B.t. hybrids are active against pests susceptible to B.t. kurstaki strains as well as against pests susceptible to B.t. tenebrionis strains. Generally, these hybrids have useful insecticidal properties which are superior to those observed by physical mixtures of the parent strains in terms of level of insecticidal activity, or in terms of spectrum of activity, or both. The insecticidal compositions comprising such microorganisms may be used to combat insects by applying the hybrids in an insecticidally effective amount to the insects or to their environment.
Another derivation from the bacterium B.t. was disclosed in European Patent Application, Publication No. 0 325 400 A1, issued to Gilroy and Wilcox. This invention relates to a hybrid toxin gene which is toxic to lepidopteran insects. Specifically, the invention comprises a hybrid delta-endotoxin gene comprising part of the B.t. var. kurstaki HD-73 toxin gene and part of the toxin gene from B.t. var. kurstaki strain HD-1. The hybrid toxin gene (DNA) encoding a protein having activity against lepidopteran insects was disclosed.
The bacterium B.t. was also utilized for its insecticidal properties in European Patent Application, Publication No. 0 340 948, issued to Wilcox, et al. This invention concerns hybrid pesticidal toxins which are produced by the fusion of an insect gut epithelial cell recognition region of a B.t. gene to diphtheria toxin B chain to prepare a hybrid B.t. toxin which is active against lepidopteran insects. It was suggested that the hybrid B.t. gene may be inserted into a plant or cloned into a baculovirus to produce a toxin which can be recovered. Alternatively, the host containing the hybrid B.t. gene can be used as an insecticide by direct application to the environment of the targeted insect.
In the search for insecticidal compounds, scorpion venom was identified as a possible source of compounds providing insecticidal properties. Two insect selective toxins isolated from the venom of the scorpion Leiurus quinquestriatus quinquestriatus were revealed in Zlotkin, et al., "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site," Arch Biochem and Biophysics, 240:877-87 (1985). In a study related to their chemical and pharmacological properties, it was revealed that one toxin induced fast excitatory contractive paralysis of fly larvae and the other induced slow depressant flaccid paralysis. Both affected sodium conductance.
Canadian Patent 2,005,658 issued Jun. 19, 1990 to Zlotkin, et al., discloses an insecticidally effective protein derived from the scorpion Leiurus quinquestriatus hebraeus. In this invention, the venom is lyophilized and separated into fractions. The fraction with the highest toxicity to larvae and the lowest toxicity to mice was subjected to further purification and the final product is that referred to as "LqhP35".
Grishin, "Toxic components from Buthus eupeus and Lycosa singoriensis venoms," Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow 117988, GSP-1, USSR, discloses four toxins isolated from the venom of the scorpion Buthus eupeus which are toxic to insects. Also disclosed was the isolation and characterization of the toxic component of the venom of the tarantula Lycosa singoriensis. The crude venom was nontoxic to insects.
Corresponding with the research and developments related to various compositions having insecticidal properties, researchers worked to develop methods for producing insecticidal genes and introducing these to a targeted pest. U.S. Pat. No. 4,879,236, issued Nov. 7, 1989 to Smith and Summers, relates to a method for incorporating a selected gene coupled with a baculovirus promoter into a baculovirus genome to produce a recombinant baculovirus expression vector capable of expression of the selected gene in an insect cell. The method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter. To prepare a recombinant transfer vector, the DNA fragment is inserted into a cloning vehicle and then a selected gene is inserted into this modified cloning vehicle such that it is under the control of the polyhedrin promoter. The recombinant transfer vector is then contacted with wild type baculovirus DNA so as to effect homologous recombination and incorporation of the selected gene into the baculovirus genome. The baculovirus Autographa californica (AcMNPV) and its associated polyhedrin promoter were found to be useful in producing a viral expression vector capable of extremely high levels of expression of a selected gene in a eukaryotic host cell.
The inventors suggest that the expression vector might be used in a system for controlling insects by selecting a gene which produces a protein which is toxic to a specific insect or to a spectrum of insects and cloning that gene into the AcMNPV expression vector. They suggest that the vector could be applied to the plant or animal to be protected. The recombinant virus could invade the cells of the intestinal wall following ingestion by the insect and begin replication. An alternative suggestion is to insert the gene into the baculovirus genome so that it would be fused to the polyhedrin structural sequence in such a way that the polyhedron coating would be dissociated by the alkaline conditions of the insect gut and the toxic product would be released.
A further method for producing insecticidal genes and introducing them to the target to be protected was disclosed in Cutler, "Electroporation: Being Developed to Transform Crops: Success with Model Crop Confirmed," AG Biotech. News vol. 7(5):3 & 17 (1990). This article teaches that DNA may be electroporated directly into germinating pollen and that pollen may be put back on the flower to form seeds which then grow into transformed plants. This method has been employed successfully in tobacco plants and may be successful in corn and alfalfa as well. This method may be easier than the electroporation of protoplasts because the ultimate goal is to pollinate the flowers and "let the flowers do the work" rather than to regenerate the plant. The process consists of collecting pollen, germinating it in a germinating medium for 30-60 minutes after which the pollen tube will start to come out of the pollen grain, adding the desired DNA to the liquid suspension containing the pollen, administering an electric shock to open pores in the pollen tube, washing the excess DNA away, and putting the altered pollen on the stigma of a plant and waiting until seeds are formed. This may be an easy method to move any gene into crop plants.
An additional delivery system was disclosed in U.S. Pat. No. 4,861,595 issued Aug. 29, 1989 to Barnes and Edwards. This invention concerns the use of treated, substantially intact, microbial cells as a delivery system of protein compounds to animals and humans. The microbial cells initially produce a protein intracellularly via a homologous gene. The protein-producing microbe is treated by chemical or physical means while the cell is substantially intact. Manipulation of the treatment process produces a nonproliferative treated microbial cell without significant loss of the activity of the intracellular compound. Since the cell will not replicate and will have a stable cell wall which may then be broken down in a desired area of the digestive system of the animal or human, it allows the timed or targeted release of the products encapsulated by the subject invention. After suitable treatment, the protein-producing microbial cell itself is used as the delivery system so no purification of the produced compound is necessary. Any protein, polypeptide, amino acid, or compound, including insecticides, that may be produced by microbial means may be the starting material of the invention.
The possibility of using DNA technology to incorporate a synthetic gene which encodes a neurotoxin found in scorpion venom was explored in Carbonell, et al., "Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors," Gene 73:409-18 (1988) This article teaches the possibility of using DNA technology to incorporate a synthetic gene which encodes a neurotoxin found in the venom of the scorpion, Buthus eupeus, into the baculovirus genome to improve baculovirus pesticides. Three methods of expression using the polyhedron promoter-based AcMNPV expression system to effect toxin production were studied. Expression of the 36 codon gene alone provided minuscule production of the toxin. Some success was found with the attachment of a signal peptide to the toxin. Significant levels of protein were produced when the toxin gene was fused to the N-terminus of polyhedron gene. However, production was ten to twenty-fold less than that observed for polyhedron itself. The limitation to expression was not believed to be at the level of transcription but at the post-transcriptional level including translation and protein stability. Paralytic activity of the toxin products was not detected.
European Patent Application, publication number 0 431 829, discloses transgenic plants which effectively express in their cells an insect-specific toxin of an insect predator in an amount sufficient so as to cause toxicity to selective insects ingesting the plant tissues. The particular toxin described was isolated from the venom of the scorpion Androctonus australis.
Researchers have also been able to isolate toxins extracted from the venom of spiders. Geren, "Neurotoxins and Necrotoxins of Spider Venoms," J. Toxicol.-Toxin Reviews 5(2):161-170 (1986), reviews work related to neurotoxins and necrotoxins and suggests that spider venom molecules may provide models for specific insecticides.
U.S. Pat. No. 4,925,664 issued to Jackson and Parks on May 15, 1990, discloses methods of treating heart and neurological diseases by applying toxins derived from the spiders Agelenopsis aperta and Hololena curta. The toxins are also effective as specific calcium channel or excitatory amino acid receptor blockers that may be used against insects and related pests.
European Patent Application, publication number 0 395 357, discloses polyamines and polypeptides isolated from the venom of the spider Agelenopsis aperta. The polyamines antagonize excitatory amino acid neurotransmitters. The polypeptides and one of the polyamines block calcium channels in living cells of various organisms. The use of said calcium channel blockers in the control of invertebrate pests is suggested.
European Patent Application, publication number 0 374 940, discloses toxins isolated from the venom of the spider Hololena curta. The polypeptides are useful as insecticides and in pharmaceuticals, for example, as calcium channel and glutamate antagonists.
Bowers, et al., "Identification and purification of an irreversible presynaptic neurotoxin from the venom of the spider Hololena curta," Proc. Natl. Acad Sci. 84:3506-3510 (1987), discloses a proteinaceous neurotoxin isolated from the venom of the spider Hololena curta and its inhibition of neuromuscular transmission in Drosophila larvae. The authors suggest that the toxin blocks presynaptic calcium channels in Drosophila motor neurons.
Quistad, et al., "Paralytic and Insecticidal Toxins from the Funnel Web Spider, Hololena Curta," Toxicon 29(3):329-336 (1991), describes one peptide and ten curtatoxins purified from venom of Hololena curta and the effect when injected into lepidopteran larvae.
Stapleton, et al., "Curtatoxins: Neurotoxic insecticidal polypeptides isolated form the funnel-web spider Hololena curta," J. Biol. Chem. 265(4):2054-2059 (1990), discloses three polypeptide neurotoxins isolated from the venom of the spider Hololena curta and the effect on the cricket Acheta domestica.
Quicke and Usherwood, "Extended Summaries Pesticides Group and Physicochemical and Biophysical Panel Symposium Novel Approaches in Agrochemical Research," Pestic. Sci. 20:315-317 (1987), discloses that toxins present in the venoms of parasitic wasps and in the venoms of some orb-web spiders cause rapid paralysis when injected into insects. The authors suggest that spider toxins are blockers of glutamate receptor gated, cation-selective membrane channels. The publication refers to low molecular weight toxins in Argiope and Araneus spider venoms as well as a toxin isolated from venom glands of the Joro spider, Nephila clavata.
Another study related to the properties of isolated spider venom toxins revealed the ability of low molecular weight factors isolated from funnel-web spider venoms to bind reversibly to calcium channels. WO 89/07608 issued Aug. 24, 1989 to Cherksey, et al., discloses that these active low molecular weight factors reversibly bind to calcium channels with sufficient specificity and affinity to extinguish calcium conductance in neurons and to permit isolation and purification of calcium channel structures. These venoms were found to be toxic to mammals.
Other applications of spider toxins were discussed in Jackson and Parks, "Spider Toxins: Recent Applications in Neurobiology," Ann Rev Neurosci 12:405-14 (1989). This article teaches that there is great heterogeneity in the toxins of different taxa. It recognizes that experiments have suggested species-specific properties of calcium channels and the spider venoms might provide calcium channel antagonists. The spider venoms discussed are found to affect vertebrates. The article also identifies spider venoms as possible sources of insect-specific toxins for agricultural applications.
Adams, et al., "Isolation and Biological Activity of Synaptic Toxins from the Venom of the Funnel Web Spider, Agelenopsis Aperta," in Insect Neurochemistry and Neurophysiology 1986, Borkovec and Gelman eds., Humana Press, New Jersey, 1986, teaches that multiple peptide toxins which antagonize synaptic transmission in insects have been isolated from the spider Agelenopsis aperta.
U.S. Pat. No. 4,855,405 issued Aug. 8, 1989 to Yoshioka, et al., discloses a receptor inhibitor obtained from Joro spider venom glands, and its manufacturing method. The compound has an insecticidal effect when insects contact the compound carried in a liquid or solid.
U.S. Pat. No. 4,918,107 issued Apr. 17, 1990 to Nakajima et al., relates to a compound which has glutamate receptor inhibitor activity, a process for preparing the same, and an insecticidal composition containing the same. The compound is carried in a liquid or solid carrier with a dispersing agent added and applied directly to the plant or animal to be protected. A low dosage is effective as an insecticide and has very low mammalian and fish toxicity and small adverse influence to the environment.
The use of baculoviruses as bioinsecticides has also been explored. The major deficit of wild type baculoviruses as bioinsecticides is that they are slow-acting. Larvae that ingest wild type baculoviruses generally die within 5 to 7 days. The infected larvae continue to feed during a significant portion of this time and substantial crop damage can occur.
Due to a combination of problems associated with some synthetic insecticides, including toxicity, environmental hazards, and loss of efficacy due to resistance, there exists a continuing need for the development of novel means of invertebrate control, including the development of genetically engineered recombinant baculoviruses which express protein toxins capable of incapacitating the host more rapidly than the baculovirus infection per se.